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Background  The invasive fruit pest  Drosophila suzukii  was reported for the first time in Europe and the USA in 2008 and has spread since then. The adoption of type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) as a tool for genome manipulation provides new ways to develop novel biotechnologically-based pest control approaches. Stage or tissue-specifically expressed genes are of particular importance in the field of insect biotechnology. The enhancer/promoter of the spermatogenesis-specific  beta-2-tubulin  ( β2t ) gene was used to drive the expression of fluorescent proteins or effector molecules in testes of agricultural pests and disease vectors for sexing, monitoring, and reproductive biology studies. Here, we demonstrate an improvement to CRISPR/Cas-based genome editing in  D. suzukii  and establish a sperm-marking system.    Results  To improve genome editing, we isolated and tested the  D. suzukii  endogenous promoters of the small nuclear RNA gene  U6  to drive the expression of a guide RNA and the  Ds heat shock protein 70  promoter to express  Cas9 . For comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. We demonstrate the homology-dependent repair (HDR)-based genome editing efficiency by applying a previously established transgenic line that expresses  DsRed  ubiquitously as a target platform. In addition, we isolated the  Ds_β2t  gene and used its promoter to drive the expression of a red fluorescence protein in the sperm. A transgenic sperm-marking strain was then established by the improved HDR-based genome editing.    Conclusion  The deployment of the endogenous promoters of the  D. suzukii U6  and  hsp70  genes to drive the expression of  gRNA  and  Cas9 , respectively, enabled the effective application of helper plasmid co-injections instead of preformed ribonucleoproteins used in previous reports for HDR-based genome editing. The sperm-marking system should help to monitor the success of pest control campaigns in the context of the Sterile Insect Technique and provides a tool for basic research in reproductive biology of this invasive pest. Furthermore, the promoter of the  β2t  gene can be used in developing novel transgenic pest control approaches and the CRISPR/Cas9 system as an additional tool for the modification of previously established transgenes.

Improvement and use of CRISPR/Cas9 to engineer a sperm-marking strain for the invasive fruit pest Drosophila suzukii